Immunohistochemical Analysis of Lung Tumor Markers

Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections of human and mouse lung tumor tissues as previously described [16 (link)]. Primary antibodies include rabbit polyclonal anti-human Oct4 (2750, Cell Signaling, Beverly, MA), rabbit monoclonal anti-human M-CSF (ab52864, Abcam, Cambridge, MA), and rabbit polyclonal anti-human/mouse CD206 (ab64693, Abcam). The immunoreactive intensity was analyzed with the HistoQuest analysis software (TissueGnostics, Vienna, Austria). Immunoblot analysis was performed to detect Oct4 and β-actin (as the loading control) using rabbit polyclonal anti-human Oct4 antibody (2750, Cell Signaling) and mouse monoclonal anti-β-actin-peroxidase antibody (A3854, Sigma-Aldrich, St. Louis, MO), respectively. For ELISA, A549 cells (1 × 10⁶) that had been transduced with recombinant lentiviruses derived from pSin-null, pSin-EF2-Oct4, shLuc, shOct4#4880, or shOct4#4883 lentiviral vectors were cultured in 6-well plates, and their conditioned media were collected after 72 h for quantifying the levels of M-CSF, TNF-α, IL-1β, and IL-6 secretion with ELISA kits (R &D, Minneapolis, MN).

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Lu C.S., Shiau A.L., Su B.H., Hsu T.S., Wang C.T., Su Y.C., Tsai M.S., Feng Y.H., Tseng Y.L., Yen Y.T., Wu C.L, & Shieh G.S. (2020). Oct4 promotes M2 macrophage polarization through upregulation of macrophage colony-stimulating factor in lung cancer. Journal of Hematology & Oncology, 13, 62.

Publication 2020

ab52864 ab64693 HistoQuest analysis software anti-β-actin-peroxidase antibody A3854 ELISA kits

A549 cells Actin Anti antibody Antibodies Conditioned media Elisa Embedded paraffin Formalin Human Il 1β Immunoblot analysis Lentiviruses Lung tumor M csf Monoclonal antibody Mouse Oct4 Peroxidase Rabbit Secretion Tissues Tnf α Vectors

Corresponding Organization : Chest Hospital, Ministry of Health and Welfare

Other organizations : National Cheng Kung University, Taipei Medical University, Chi Mei Medical Center

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Variable analysis

independent variables

Recombinant lentiviruses derived from pSin-null, pSin-EF2-Oct4, shLuc, shOct4#4880, or shOct4#4883 lentiviral vectors

dependent variables

Levels of M-CSF, TNF-α, IL-1β, and IL-6 secretion
Immunoreactive intensity

control variables

A549 cells (1 × 10^6) cultured in 6-well plates
Conditioned media collected after 72 h

controls

Positive control: Not specified
Negative control: pSin-null lentiviral vector

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